The Toxicity Of Mono (2-Ethylhexyl) Phthalate On Primary Cultured Hypothalamic Neurons Of Neonatal Rats

ObjectiveHypothalamus,which is an important endocrine gland to control the neuroendocrine system,is very sensitive to endogenous and exogenous hormones in critical period of its development.Mono(2-ethylhexyl)phthalate(MEHP),one of di-2-ethylexyl phthalate(DEHP)metabolites,has anti-androgenic properties and estrogenic properties.MEHP may potentially interfere with the development of hypothalamic neurons,but few relevant studies have been reported.The aims of this study is to explore the effects of MEHP on the development of neonatal rats’hypothalamus neurons in vitro and the dose-response relationship model of its toxicity.The pathway and mechanisms of MEHP’ effects on hypothalamus neurons were studied by examining the neurons apoptosis induced by MEHP,as well as its antagonistic effects on E2/T mediated proliferation and anti-apoptosis of hypothalamic neurons.Methods(1)The primary culture and identification of the hypothalamic neurons of neonatal rats:in the primary culture of neurons,single cell suspension was prepared using the improved mechanical methods.After culture in DMEM supplement with 10%Charcoal/Dextran-Fetal Bovine Serum for 24h,the medium was replaced with serum-free medium supplement with 97%Neurobasal,2%B27 and 1%Glutamine to inhibit excessive proliferation of glial cells.Neuron-specific enolase(NSE)was used to identify the hypothalamic neurons.The morphological characteristics of hypothalamic neurons was observed using HE staining and Nissl staining methods.(2)The 7d cultured hypothalamic neurons were treated 24h or 48h in different concentrations of MEHP(10-15mol/L～10-3mol/L.MTT method was used to determine absorbance values and cell viability and inhibition rate was calculated.Dose-response model of MEHP ’ s effects on development of hypothalamic neurons was explored by measuring the neuronal neurite length,the soma areas and the number of ridges.The effects of MEHP on the growth of neurons was observed.(3)The neuronal apoptosis of different MEHP concentration treatment group was observed via Hoechst 33258 fluorescent staining.After treated neurons with different concentrations of E2/T,absorbance values were determined by MTT method and the suitable concentration of E2/T was selected for the subsequent study.After treatment neurons with different concentrations of MEHP combined with 10-7mol/L E2/T,the neuronal neurite lengths,the soma areas and the number of ridges were measured,and the MEHP and E2/T combined effects on the growth of neurons were studied.Furthermore,Hoechst 33258 fluorescent staining was used to study the morphological changes of cellular nucleus and neuronal apoptosis under effects of MEHP itself and MEHP combined E2/T.Results(1)In thhis study,the hypothalamic neurons grow well and the purity of the neurons can reach 95%and above.(2)After treated with different concentrations MEHP for 24h,the refraction of neurons decreased,the soma area shrank,the neuronal neurite shortened and the connections between neurons fractured among the concentrations of 10-15mol/L、10-13mol/L、10-11mol/L、10-9mol/L、10-7mol/L、10-5mol/L、10-3mol/L MEHP.The inhibition rate of 10-15 mol/L and 10-3 mol/L MEHP were 30.1%and 33.6%,respectively,which inhibited the growth of neurons significantly.However,the inhibition rate of 10-9mol/LMEHP was 1.6%and the inhibitory effect was not obvious.The inhibition rate and dose-response relationship of different MEHP concentrations treated hypothalamic neurons suggested a U-shape relationship.(3)The chromatin of neurons wrinkled,the shape showed irregular and the apoptotic bodies appeared.The highest apoptosis rate was 83.9%of concentration 10-3mol/L MEHP The apoptosis rate of MEHP-treated groups increased compared with the control,and the difference was statistically significant(P<0.05).(4)Estradiol(E2)/testosterone(T)treatment promoted the growth of neurons in all concentration group.The concentration of 10-7molE2/T was selected for the trial.(5)The neurites and soma area of MEHP combined E2/T(10-7mol/L)treated hypothalamic neurons decreased to different levels.The inhibition of neurite length was(20.14±3.60)μm and(21.8 ± 2.07)μm in the 10-3mol/L MEHP + 10-7mol/L E2/T treated group,which is the most significant.Compared with E2/T group,MEHP antagonized the effect of E2/T mediated proliferation of hypothalamic neurons.(6)The apoptosis rates of 10-7mol/L,10-5mol/L,10-3mol/L MEHP combined with 10-7mol/L E2/T were 27.97%,42.8%,68.66%,respectively.The differences between the treatment group and the E2/T group were statistically significant(P<0.05).MEHP antagonized the anti-apoptotic effect of E2/T-mediated hypothalamic neurons.ConclusionIn this study,MEHP inhibited the proliferation of hypothalamic neurons.It suggested that the effect of MEHP on hypothalamus neurons was non-monotonic dose-response relationship.MEHP had direct apoptotie effect on the neurons.MEHP antagonized the proliferation and anti-apoptotic effects of E2/T-mediated hypothalamic neurons.The mechanism of MEHP’s effects on the neurons remained to be further studied.